PROJECT SUMMARY Grp94 is an ER HSP90 chaperone whose broad oncogenic roles have only recently been established. However, understanding of grp94 structure and mechanism has lagged behind that of other HSP90s. grp94 exhibits high sequence and structural homology to the other cellular HSP90 paralogs. Nevertheless, attempts to demonstrate complementation between the cytosolic and ER paralogs have consistently failed in both directions, suggesting that the enormous metabolic burden of maintaining high levels of a specialized ER- specific HSP90 has a distinct mechanistic purpose. We propose that this distinct mechanism is reflected in its structure. The goal of this project is to understand what makes grp94 unique in the HSP90 family and to exploit these features in the design of new inhibitors as cancer therapeutics. We will address this question in four specific aims. The first is to characterize and develop next-generation grp94-selective inhibitors. Together with Dr. Gabriela Chiosis, leader of Project 2, we have characterized a set of purine-based (PU) compounds that bind selectively to grp94 and exhibit anti-cancer activity. Higher affinity and more selective compounds are needed to improve their therapeutic potential and to serve as chemical probes of grp94 function. The second is to determine the structural basis for selective binding of PU ligands to grp94. We will investigate how the conformational changes in grp94 that foster selective inhibitor binding manage to occur in one paralog and not another ? what specific sequence and structural elements make the grp94 response distinct? Third, we aim to determine how individual structural domains contribute to the unique biochemical and functional properties of grp94. In collaboration with Dr. Zihai Li, leader of Project 1, we will examine how individual grp94 domains contribute to the overall specialization and function of this paralog as it matures its client proteins such as GARP. Fourth, we will probe the role of post-translational modifications (PTMs) on grp94 client binding and activity. Together these experiments should pave the way for the development of new anti-cancer therapeutics and illuminate central mechanistic questions about grp94 action.